Xiao-Yi Qi

Curcumin Inhibits Melanogenesis in Human Melanocytes

Plant derived compounds, as potentially safe and effective skin lightening agents (SLAs), have attracted great attention from many researchers. Curcumin is a plant‐derived polyphenol, which has been reported to suppress melanogenesis in B16 melanoma cells. However, little is known about whether curcumin affects melanogenesis in cultured human melanocytes. In addition, the molecular mechanism for the antimelanogenic effects of curcumin remains largely unknown. The present study assessed the effects of curcumin on melanin synthesis, cellular tyrosinase activity, the expression of melanogenesis‐related proteins (microphthalmia‐associated transcription factor (MITF), tyrosinase, tyrosinase‐related protein 1 and 2 (TRP‐1, TRP‐2)), and activation of melanogenesis‐regulating signals including phosphatidylinositol 3‐kinase (PI3K)/Akt/ glycogen synthase kinase 3 (GSK 3β), extracellular signal‐regulated kinase (ERK) and p38 MAPK in human melanocytes. The results showed that the melanin content and tyrosinase activity, as well as the expression of melanogenesis‐related proteins in human melanocytes, were significantly inhibited by curcumin in a dose dependent manner. In addition, PI3K/Akt/ GSK 3β, ERK and p38 MAPK were activated by curcumin, while inhibitors of these signals attenuated the inhibitory effects of curcumin on melanogenesis. These results suggest that curcumin inhibits melanogenesis in human melanocytes through activation of Akt/GSK 3β, ERK or p38 MAPK signaling pathways. Copyright

Targeting RAS/RAF/MEK/ERK signaling in metastatic melanoma

The RAS/RAF/MEK/ERK pathway has been reported to be activated in over 80% of all cutaneous melanomas, making it the focus of many scientific studies in the melanoma field. Discoveries of mutations and aberrant expression of components in this cascade, in particular, BRAF and NRAS render a deeper understanding of the mechanisms responsible for oncogenesis and provide new therapeutic strategies for this deadly disease. This review starts with a comprehensive discussion on the role of this pathway in initiation and progress of melanoma. Mechanistically, mutated BRAF and NRAS exert most of the oncogenic effects through the activation of the MAPK pathway, which both drive the uncontrolled growth of melanoma cells and regulate the cell survival. In a subsequent section, clinical efficacy of targeted small‐molecule inhibitors is highlighted. BRAF‐targeted therapies (e.g., vemurafenib, dabrafenib) have showed impressive results in systemic therapy for melanoma harboring activating BRAF V600E mutations. MEK inhibitors show limited activity in phase I trials, and inhibitors directly targeting mutated NRAS, to date, have not been realized. Furthermore, the emerging mechanisms underlying both intrinsic and acquired drug resistance as well as approaches to prevent or abrogate the onset of therapeutic escape are addressed. Finally, the promising vistas and major challenges involving small‐molecule inhibitors targeting this MAPK pathway in melanoma therapy are briefly discussed. It can be envisaged that disseminated melanoma is no longer such a bleak prognosis in future given the research and development of new signal transduction inhibitors based on our evolving understanding of melanoma genetics and intracellular signaling.

Inhibitory effects of sanguinarine on human liver cytochrome P450 enzymes

Sanguinarine (SAG) has been recognized as an anticancer drug candidate. However, the drug-drug interactions (DDI) potential for SAG via the inhibition against human cytochrome P450 (CYP) enzymes remains unclear. In the present study, the inhibitory effects of SAG on seven major human CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C8, 2C9 and 3A4 were investigated with human liver microsomes (HLM). The results showed that SAG was a potent noncompetitive inhibitor of CYP2C8 activity (Ki=8.9 μM), and competitive inhibitor of CYP1A2, CYP2C9 and CYP3A4 activities (Ki=2.7, 3.8 and 2.0 μM, respectively). Furthermore, SAG exhibited time- and NADPH-dependent inhibition towards CYP1A2 and CYP3A4 with KI/kinact values of 13.3/0.087 and 5.58/0.029 min(-1) μM(-1), respectively. Weak inhibition of SAG against CYP2E1, CYP2D6 and CYP2A6 was also observed. In vitro-in vivo extrapolation (IV-IVE) from HLM data showed that more than 35.9% of CYP1A2, CYP2C9, CYP2C8 and CYP3A4 activities in vivo could be inhibited by SAG, suggesting that harmful DDIs could occur when SAG or its medical preparations are co-administered with drugs primarily cleared by these CYP isoforms. Further in vivo studies are needed to evaluate the clinical significance of the data presented herein.

Characterization of circulating CD8+T cells expressing skin homing and cytotoxic molecules in active non-segmental vitiligo.

BACKGROUND Vitiligo is caused by melanocyte depletion. Studies have suggested that skin-homing cytotoxic T lymphocytes that express cutaneous lymphocyte-associated antigen (CLA) are responsible for melanocyte depletion. The characteristics of these skin-homing cytotoxic T cells have not been well established yet. OBJECTIVES To investigate the frequency of skin-homing CD8(+)T cells (CD8(+)CLA(+)T cells) and their expression of cytotoxic molecules, as well as migration-related molecules in CD8(+)T cell in non-segmental vitiligo patients. MATERIALS & METHODS The frequency of CD8(+)CLA(+)T cells and their expression of cytotoxic molecules (perforin, granzyme-B and FasL) in peripheral blood of patients with non-segmental vitiligo were assessed using flow cytometry. Levels of chemokine receptors (CCR4, CCR10) on CD8(+)T cells were evaluated. RESULTS Our results revealed a higher frequency and increased expression of perforin and granzyme-B in circulating CD8(+)CLA(+)T cells from patients with active vitiligo. The expression levels of CCR4 increased in CD8(+)T cells in active vitiligo patients. CONCLUSION Patients with active non-segmental vitiligo have a higher frequency of CD8(+)CLA(+)T cells and hyper-activated cytotoxic functions, which may be involved in the pathogenesis of non-segmental vitiligo.

Apigenin attenuates dopamine-induced apoptosis in melanocytes via oxidative stress-related p38, c-Jun NH2-terminal kinase and Akt signaling

BACKGROUND Accumulating evidence suggests that the occurrence of oxidative stress leads to melanocyte degeneration in vitiligo. Elevated level of dopamine (DA), an initiator of oxidative stress, reportedly is found in patients with vitiligo and induces melanocyte death in vitro. DA-treated melanocytes have been used as a model to search for antioxidants for treating vitiligo. OBJECTIVE We investigated the protective effects of apigenin against DA-induced apoptosis in melanocytes and the molecular mechanism underlying those effects. METHODS Melanocytes with or without pretreatment with apigenin were exposed to DA. Then cell viabilities were measured by MTT assay. Cellular reactive oxygen species (ROS) levels and the percentage of apoptotic cells were detected by flow cytometry analysis. Activation of caspase 3, poly(ADP-ribose) polymerase (PARP) and oxidative stress-related signaling, including p38, c-Jun NH2-terminal kinase (JNK) and Akt, were assessed by Western blotting. RESULTS Apigenin attenuated DA-induced apoptotic cell death, relieved ROS accumulation and activated caspase 3 and PARP, suggesting the protective effects of apigenin against DA-induced oxidative stress and apoptosis in melanocytes. Moreover, DA induced phosphorylation of p38, JNK and Akt, while inhibitors of p38, JNK and Akt significantly decreased DA-induced apoptosis. However, pretreatment with apigenin significantly inhibited DA-triggered activation of p38, JNK and Akt, suggesting the involvement of p38, JNK and Akt in the protective effects of apigenin against DA-induced cytotoxicity. CONCLUSION These results suggest that apigenin attenuates dopamine-induced apoptosis in melanocytes via oxidative stress-related p38, JNK and Akt signaling and therefore could be a potential agent in treating vitiligo.

Identifying and applying a highly selective probe to simultaneously determine the O -glucuronidation activity of human UGT1A3 and UGT1A4

Glucuronidation mediated by uridine 5′-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway. However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme. In this paper, desacetylcinobufagin (DACB) 3-O- and 16-O-glucuronidation were found to be isoform-specific probe reactions for UGT1A4 and UGT1A3, respectively. DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

Methylation, Glucuronidation, and Sulfonation of Daphnetin in Human Hepatic Preparations In Vitro: Metabolic Profiling, Pathway Comparison, and Bioactivity Analysis

Our previous study demonstrated that daphnetin is subject to glucuronidation in vitro. However, daphnetin metabolism is still poorly documented. This study aimed to investigate daphnetin metabolism and its consequent effect on the bioactivity. Metabolic profiles obtained by human liver S9 fractions and human hepatocytes showed that daphnetin was metabolized by glucuronidation, sulfonation, and methylation to form 6 conjugates which were synthesized and identified as 7-O-glucuronide, 8-O-glucuronide, 7-O-sulfate and 8-O-sulfate, 8-O-methylate, and 7-O-suflo-8-O-methylate. Regioselective 8-O-methylation of daphnetin was investigated using in silico docking calculations, and the results suggested that a close proximity (2.03 Å) of 8-OH to the critical residue Lysine 144 might be the responsible mechanism. Compared with glucuronidation and sulfonation pathways, the methylation of daphnetin had a high clearance rate (470 μL/min/mg) in human liver S9 fractions and contributed to a large amount (37.3%) of the methyl-derived metabolites in human hepatocyte. Reaction phenotyping studies showed the major role of SULT1A1, -1A2, and -1A3 in daphnetin sulfonation, and soluble COMT in daphnetin 8-O-methylation. Of the metabolites, only 8-O-methyldaphnetin exhibited an inhibitory activity on lymphocyte proliferation comparable to that of daphnetin. In conclusion, methylation is a crucial pathway for daphnetin clearance and might be involved in pharmacologic actions of daphnetin in humans.

Regulatory T cells from active non-segmental vitiligo exhibit lower suppressive ability on CD8 + CLA + T cells

BackgroundRecent studies have shown that vitiligo is a T-cell mediated autoimmune disease. Skin-homing cytotoxic T lymphocytes expressing cutaneous lymphocyte-associated antigen (CLA) have been suggested to be responsible for the destruction of melanocytes in vitiligo. An aberration in the suppressive function of regulatory T cells (Tregs) has been reported in vitiligo patients. However, whether the weakened suppressive ability of the Tregs contributes to hyper-activated skin homing CD8+CLA+ T cells remains to be determined.ObjectivesTo investigate the inhibition of circulating Tregs on the proliferation of autologous CD8+CLA+ T cells in non-segmental vitiligo patients.MethodsCD8+CLA+ T cells and Tregs were obtained from the peripheral blood of 13 non-segmental vitiligo patients and 7 controls. The proliferative responses of CD8+CLA+ T cells were assessed in the absence or presence of autologous Tregs, and the levels of Transforming Growth Factor β1(TGF-β1) and IL-10 in culture supernatants were detected by enzyme-linked immunosorbent assay.ResultsThe proliferative responses of circulating CD8+CLA+ T cells in the presence of Tregs were significantly higher in the active vitiligo than in the stable vitiligo and control groups. Tregs from active vitiligo patients exhibited a lower inhibitory effect on proliferation of CD8+CLA+ T cells. The levels of TGF-β1 produced by Tregs were significantly lower in active vitiligo than other groups and anti-TGF-β1 antibodies could abrogate the suppressive function of Tregs.ConclusionsThe functional activity of Tregs is compromised in active vitiligo patients. TGF-β1 plays an important role in the autoimmune mechanism of the disease.

In Vitro Evaluation of the Effect of 7-Methyl Substitution on Glucuronidation of Daphnetin: Metabolic Stability, Isoform Selectivity, and Bioactivity Analysis

The C-8 phenol group is essential to exert the bioactivities of daphnetin, but it is readily conjugated with glucuronic acid prior to excretion. In this study, daphnetin-7-methylether (7M-DNP) was used to investigate the effect of 7-methyl substitution on daphnetin glucuronidation in human/rat liver (HLM/RLM) and intestine (HIM/RIM) microsomes, and recombinant UDP-glucuronosyltransferases (UGTs). Compared with daphnetin, the Vmax /Km values of 7M-DNP via 8-O-glucuronidation were 2.1-fold lower in HLM, 1.7-fold lower in HIM, and 2.4-fold lower in RLM, suggesting an improvement in metabolic stability. Different from daphnetin 8-O-glucuronidation exclusively catalyzed by UGT1A6 and UGT1A9, UGT1A1, -1A3, -1A7, -1A8, and -1A9 showed glucuronidation activity toward 7M-DNP. Kinetics studies, chemical inhibition, and the relative activity factor approach were used to demonstrate that UGT1A9 was mainly responsible for the reaction in HLM, whereas UGT1A1 was a primary contributor in HIM. The Vmax /Km values of 7M-DNP glucuronidation in HLM and HIM were 0.61-0.74-fold lower than those of rat, suggesting the differences between the two species. The bioactivity analysis demonstrated that 7M-DNP had an anti-inflammatory activity comparable to that of daphnetin. These findings indicated that the outcomes of 7-methyl substitution on daphnetin might be positive, but this should be confirmed in future in vivo studies.

Rapid profiling and target analysis of principal components in Fuling Decoctions by UFLC-DAD-ESI-MS

The traditional Chinese medicine formula Fuling Decoction (FD) has been clinically used for eczema treatment, but the unclear chemical distribution and the lack of quality control have strongly restricted its application. In this study, an analytical method incorporating ultra-fast liquid chromatography (UFLC) with MS and UV detection was developed for rapid profiling of the chemical constitutes from FD. Fourteen constitutes were identified by UFLC-ESI-MS, while four major components including genipingentiobioside, geniposide, paeoniflorin and liquiritin were quantified simultaneously by UFLC-DAD. The UFLC-based method was fully validated and can be applied to screening and determination of principal components in commercially FD prescriptions.